The Plant Cell, Vol. 24: 2515–2527, June 2012, www.plantcell.org ã 2012 American Society of Plant Biologists.

All rights reserved.

Auxin Controls Arabidopsis Adventitious Root Initiation

by Regulating Jasmonic Acid Homeostasis W

Laurent Gutierrez,a,b,1 Gaëlle Mongelard,b,1 Kristýna Floková,c Daniel I. Păcurar,d Ondrej Novák,a,c Paul Staswick,e
Mariusz Kowalczyk,a Monica Păcurar,a,f Hervé Demailly,b Gaia Geiss,a and Catherine Bellinid,g,2
a Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences,

90183 Umea, Sweden

b Centre de Ressources Régionales en Biologie Moléculaire, Université de Picardie Jules Verne, 80039 Amiens, France
c Laboratory of Growth Regulators, Faculty of Science, Palacký University and Institute of Experimental Botany, Academy of Sciences

of the Czech Republic, 78371 Olomouc, Czech Republic

d Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, 90187 Umea, Sweden
e Department of Agronomy and Horticulture, University of Nebraska, Lincoln, Nebraska 68583-0915
f University of Agricultural Sciences and Veterinary Medicine, 400372 Cluj Napoca, Romania
g Institut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318 Institut National de la Recherche Agronomique–AgroParisTech, Institut

National de la Recherche Agronomique Centre de Versailles–Grignon, F-78026 Versailles cedex, France

Vegetative shoot-based propagation of plants, including mass propagation of elite genotypes, is dependent on the development
of shoot-borne roots, which are also called adventitious roots. Multiple endogenous and environmental factors control the
complex process of adventitious rooting. In the past few years, we have shown that the auxin response factors ARF6 and ARF8,
targets of the microRNA miR167, are positive regulators of adventitious rooting, whereas ARF17, a target of miR160, is a negative
regulator. We showed that these genes have overlapping expression profiles during adventitious rooting and that they regulate
each other’s expression at the transcriptional and posttranscriptional levels by modulating the homeostasis of miR160 and
miR167. We demonstrate here that this complex network of transcription factors regulates the expression of three auxin-
inducible Gretchen Hagen3 (GH3) genes, GH3.3, GH3.5, and GH3.6, encoding acyl-acid-amido synthetases. We show that these
three GH3 genes are required for fine-tuning adventitious root initiation in the Arabidopsis thaliana hypocotyl, and we
demonstrate that they act by modulating jasmonic acid homeostasis. We propose a model in which adventitious rooting is an
adaptive developmental response involving crosstalk between the auxin and jasmonate regulatory pathways.

INTRODUCTION involved in the regulation of adventitious rooting have been

Adventitious roots arise from aerial organs of the plant either
naturally or in response to altered environmental conditions
(Geiss et al., 2009; Li et al., 2009). They are required for vege-
tative propagation of plants and the successful clonal multipli-
cation of elite genotypes of forest, horticultural, and agricultural
plant species. The ability to form adventitious roots is a heritable
quantitative trait controlled by multiple endogenous and envi-
ronmental factors (Geiss et al., 2009; Li et al., 2009), among
which the plant hormone auxin plays a central role. Auxin is
often applied exogenously to promote the development of ad-
ventitious roots on stem cuttings of difficult-to-root genotypes.
However, exogenous auxin is not always effective, and a better
understanding of the molecular mechanisms through which it
regulates adventitious rooting is needed in order to improve
the rooting of recalcitrant genotypes. So far, only a few genes
1 These authors contributed equally to this work.
2 Address correspondence to catherine.bellini@plantphys.umu.se.
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Catherine Bellini
(catherine.bellini@plantphys.umu.se).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.112.099119
identified. In rice (Oryza sativa), disruption of the auxin-inducible
CROWN ROOTLESS1/ADVENTITIOUS ROOTLESS1 gene (CRL1/
ARL1), which encodes a member of the plant-specific LATERAL
ORGAN BOUNDARY DOMAIN (LBD) protein family, has been
shown to prevent the initiation of adventitious crown root primordia
(Inukai et al., 2005; Liu et al., 2005). The promoter of the CRL1/
ARL1 gene contains specific cis-regulatory elements that interact
with the rice transcription factor ARF16, a member of the AUXIN
RESPONSE FACTOR (ARF) family (Inukai et al., 2005). ARF16 is
the rice ortholog of both ARF7 and ARF19 in Arabidopsis thaliana
(Wang et al., 2007). ARF7 and ARF19 regulate lateral root forma-
tion in Arabidopsis by activating the expression of LBD16 and
LBD29, both of which are phylogenetically closely related to ARL1/
CRL1 (Wilmoth et al., 2005; Okushima et al., 2007). ARF tran-
scription factors mediate auxin signaling at the transcriptional level
by regulating the expression of auxin-responsive genes (Guilfoyle
and Hagen, 2007). The characterization of Arabidopsis argonaute1
(Bohmert et al., 1998) and superroot2 (Delarue et al., 1998; Barlier
et al., 2000) single and double mutants allowed us to identify sev-
eral genes potentially involved in the regulation of adventitious
rooting, including one ARF transcription factor, ARF17 (At1g77850),
that encodes the target of the microRNA miR160 and three auxin-
inducible GH3-like genes, GH3.3 (At2g23170), GH3.5/GH3a/WES1
(At4g27260), and GH3.6/DFL1 (At5g54510; Nakazawa et al., 2001;
2516 The Plant Cell

Tanaka et al., 2002; Sorin et al., 2005, 2006; Park et al., 2007b,
2007a). The first GH3 gene was identified as an early auxin-
responsive gene in soybean (Glycine max) (Hagen et al., 1984),
and since then, GH3-like genes have been found in many plant
species. However, it is only recently that they have been shown
to encode enzymes able to conjugate various amino acids to
jasmonates, auxins, and benzoates, leading to the activation,
inactivation, or degradation of these molecules (Staswick et al.,
2005; Chen et al., 2010; Wang et al., 2010; Westfall et al., 2010).
We first showed that ARF17 was a negative regulator of ad-
ventitious rooting that could potentially integrate auxin and light
signaling pathways affecting this process (Sorin et al., 2005). We
also showed that ARF17 was a negative regulator of GH3.3,
GH3.5, and GH3.6 expression (Sorin et al., 2005), and, in addition,
we found that the GH3.3, GH3.5, and GH3.6 protein content was
positively correlated with the number of adventitious roots but not
with the endogenous auxin content (Sorin et al., 2006). More re-
cently, we demonstrated that adventitious root initiation is con-
trolled in Arabidopsis hypocotyls by a subtle balance between the
negative regulator ARF17, which is the target of miR160, and the
positive regulators ARF6 (At1g30330) and ARF8 (At5g37020),
which are targets of miR167. These three ARFs display over-
lapping expression domains, interact genetically, and regulate
each other’s expression at both the transcriptional and post-
transcriptional levels by modulating the availability of the regula-
tory microRNAs miR160 and miR167 (Gutierrez et al., 2009). In this
article, we show that, in contrast to ARF17, ARF6 and ARF8 are
positive regulators of the auxin-inducible genes GH3.3, GH3.5,
and GH3.6. We also demonstrate that these three GH3 genes
have redundant functions and are required for fine-tuning ad-
ventitious root initiation in Arabidopsis hypocotyls by modulating
jasmonic acid (JA) homeostasis.
RESULTS
The Number of Adventitious Roots in arf Mutants and
ARF-Overexpressing Lines Does Not Correlate with
Endogenous Auxin Content But Does Correlate with
GH3.3, GH3.5, and GH3.6 Transcript Levels
In an attempt to identify the pathway through which the micro-
RNA-targeted genes ARF6, ARF8, and ARF17 regulate adventi-
tious rooting, we selected for evaluation representative mutants
and overexpressing lines that we previously characterized as
having contrasting adventitious rooting phenotypes (Gutierrez
et al., 2009) (Figure 1A). The double mutant arf10-3 arf16-3, which
has no phenotype affecting adventitious root growth, was used as
a control for the MIR160c-overexpressing line (MIR160c-OX), in
which the accumulation of miR160 specifically targets ARF10,
ARF16, and ARF17 transcripts for degradation (Wang et al., 2005).
In the absence of an arf17 knockout mutant, we used MIR160-OX
as an ARF17 downregulated line, as described previously (Gutierrez
et al., 2009).
Since auxin is a well-known inducer of adventitious rooting in
Arabidopsis (Boerjan et al., 1995; Delarue et al., 1998), it was

Figure 1. Variation in the Number of Adventitious Roots in the Hypocotyl of arf Mutants and ARF-Overexpressing Lines Correlates with the Expression
Levels of GH3.3, GH3.5, and GH3.6.

(A) Adventitious roots (AR) were counted on seedlings of arf mutant lines and ARF-overexpressing lines. Seedlings were first etiolated in the dark, until
their hypocotyls were 6 mm long, and then transferred to the light for 7 d. Data from three independent biological replicates, each of at least 30
seedlings, were pooled and averaged. Error bars indicate SE. A one-way analysis of variance combined with the Tukey’s multiple comparison posttest
indicated that the values marked with asterisks were significantly different from wild-type values (P < 0.01; n > 90). In addition, those marked with hash
signs were significantly different from values obtained from single mutants or the ARF17-OX1 line (P < 0.01; n > 90). Col-0, ecotype Columbia; Ws,
ecotype Wassilewskija.
(B) The GH3.3, GH3.5, and GH3.6 transcript abundance was quantified in the hypocotyls of the different arf mutants or overexpressing lines, which had
been etiolated and then transferred to the light for 72 h. Gene expression values are relative to expression in the wild type, for which the value is set to 1.
Error bars indicate SE obtained from three independent biological replicates. A one-way analysis of variance combined with the Dunnett’s comparison
posttest indicated that the values marked with ns were not significantly different from wild-type values, whereas the others were significantly different
(P < 0.05; n = 3).

important to verify whether the increased or reduced number of
adventitious roots was due to a higher or lower endogenous con-
tent of free indole-3-acetic acid (IAA) in the hypocotyl. Therefore,
the level of endogenous free IAA and its major conjugates, IAAsp
[N-(indole-3-ylacetyl)-aspartate] and IAGlu [N-(indole-3-ylacetyl)-
glutamate], was quantified in the hypocotyls of seedlings grown as
described previously (Gutierrez et al., 2009). Neither mutations in,
nor overexpression of, ARF6, ARF8, or ARF17 significantly altered
the endogenous content of free IAA, suggesting that the observed
adventitious root phenotype is unlikely to be related to an increase
or a reduction in IAA content (see Supplemental Figure 1 online).
To investigate the interactions between GH3.3, GH3.5, and
GH3.6, whose expression levels were correlated with the num-
ber of adventitious roots (Sorin et al., 2006) and ARF genes, we
first analyzed the expression of the three GH3 genes during the
early steps of adventitious root formation using transcriptional
fusion constructs containing b-glucuronidase (GUS) fused to the
respective GH3 promoters. The promGH3.3:GUS, promGH3.5:
GUS, and promGH3.6:GUS constructs have overlapping ex-
pression patterns with those of promARF6:GUS, promARF8:
GUS, and promARF17:GUS and are likewise regulated by light
(see Supplemental Figure 2 online) (Gutierrez et al., 2009).
Second, we performed real-time quantitative RT-PCR analysis
of the relative transcript amounts of the three GH3 genes in the
hypocotyls of the various ARF mutant and overexpressing lines
(Figure 1B). In the double mutant arf10-3arf16-3, which displayed
no difference in adventitious rooting phenotype compared with
the wild type, the GH3.3, GH3.5, and GH3.6 transcript levels were
not affected. In contrast, the transcript levels of all three genes
were reduced in all of the lines showing fewer adventitious roots
than the wild type, whereas it was increased in those with a higher
number of adventitious roots (Figures 1A and 1B). In agreement
with previous data (Sorin et al., 2006), the smaller the number of
adventitious roots, the lower the amount of GH3.3, GH3.5, and
GH3.6 transcripts and vice versa. Since the arf6-3arf8-7 double
mutant is sterile, we analyzed a population of progeny from the
sesquimutant arf6-3/arf6-3 arf8-7/ARF8-7 segregating for 25%
homozygous double mutants. The average number of adventi-
tious roots was significantly lower than that of the wild type and of
the single mutants arf6-3 and arf8-7, indicating an additive effect
of the two positive regulators ARF6 and ARF8 (Figure 1A). This
additive effect was confirmed by the reduced level of the total
GH3.3+GH3.5+GH3.6 relative transcript amount in the sesquimu-
tant compared with that in the single mutants (see Supplemental
Figure 3A online). In contrast, the three GH3 genes were expressed
at a higher level in ARF6- and ARF8-overexpressing lines. The
three GH3 genes were also downregulated in the ARF17-OX1 line
(Figure 1B) (Sorin et al., 2005) and, as expected, upregulated in the
MIR160c-OX line, in which ARF17 is downregulated. When
ARF17 was overexpressed in the knockout arf6-3 and arf8-7
mutant backgrounds, a significant decrease in the relative amount
of transcript of each of the three GH3 genes was observed
compared with the ARF17-overexpressing lines or the arf6-3 and
arf8-7 single mutants (Figure 1B). This confirmed the exis-
tence of an additive effect due to the overexpression of the
negative regulator ARF17 in mutants lacking one of the pos-
itive regulators (ARF6 or ARF8) of GH3.3, GH3.5, and GH3.6
expression.
IAA and JA Control Adventitious Rooting 2517
GH3.3, GH3.5, and GH3.6 belong to a family of 19 genes.
Therefore, we checked, in the hypocotyl of the ARF mutant and
overexpressing lines, the expression of the other members of
the GH3 gene family as well as that of selected auxin-inducible
genes shown to be differentially expressed in the ago1-3 versus
wild-type hypocotyls (http://urgv.evry.inra.fr/cgi-bin/projects/
CATdb/catdb_index.pl). None of these genes were found to be
coregulated with GH3.3, GH3.5, and GH3.6 in the different ARF
mutant or overexpressing lines (see Supplemental Figures 3B and
3C online).
GH3.3, GH3.5, and GH3.6 Positively and Redundantly
Regulate Adventitious Rooting
In order to assess the potential contributions of the three GH3
genes to the regulation of adventitious rooting, we analyzed ad-
ventitious root formation in gh3.3-1, gh3.3-2, gh3.5-1, gh3.5-2,
gh3.6-1, and gh3.6-2 single knockout mutant alleles, in gh3.3-
1gh3.5-2 and gh3.3-1gh3.6-1 double mutants, and in a gh3.3-
1gh3.5-2gh3.6-1 triple mutant grown under conditions described
previously (Sorin et al., 2005; Gutierrez et al., 2009) (Figure 2A). We
confirmed that none of the single, double, or triple gh3 mutants
showed a defect in primary root elongation or secondary root
development when grown in our conditions (see Supplemental
Figure 4 online).
Interestingly, all single mutants produced significantly more
adventitious roots than did the wild type. This was unexpected,
given that the results presented above, together with our pre-
vious data (Sorin et al., 2006), indicated a positive correlation
between the number of adventitious roots and the GH3 tran-
script or GH3 protein content. This phenotype was not explained
by an increased endogenous auxin content since, despite the
fact that these three GH3 proteins were shown to conjugate IAA
(Staswick et al., 2005), no modification of the endogenous free
IAA content was observed in the single mutants (see Supplemental
Figure 5 online). In contrast to the single mutants, the gh3.3-
1gh3.5-2 and gh3.3-1gh3.6-1 double mutants developed only half
the number of adventitious roots compared with the wild type.
Also, the gh3.3-1gh3.5-2gh3.6-1 triple mutant produced signifi-
cantly fewer adventitious roots than the double mutants (Figure
2A), showing the additive effect of the mutations and suggesting
a redundant function for GH3.3, GH3.5, and GH3.6 in the regula-
tion of adventitious rooting in Arabidopsis. In this particular context,
this result was also contradictory with a role in IAA conjugation and
a potential increase of free IAA, since one would expect to see the
number of adventitious roots increasing and not decreasing in the
double and triple mutants. This did not explain why the single
mutants produced more adventitious roots than the wild type.
Because the three GH3 genes were strictly coregulated in the
different arf mutants and ARF-overexpressing lines, we performed
quantitative RT-PCR analysis of the relative transcript amounts of
these GH3 genes in the hypocotyl of each single and double gh3
mutant (Figure 2A). Interestingly, the expression of GH3.5 and
GH3.6 was upregulated in gh3.3-1 mutant seedlings, whereas that
of GH3.3 and GH3.6 was upregulated in gh3.5-2 mutant seedlings,
and that of GH3.3 and GH3.5 was upregulated in gh3.6-1 mutant
seedlings (Figure 2B). This compensatory effect led to a higher
level of total GH3.5+GH3.6, GH3.3+GH3.6, and GH3.3+GH3.5
2518 The Plant Cell
Figure 2. GH3.3, GH3.5, and GH3.6 Genes Have Redundant Functions
in the Regulation of Adventitious Rooting.
(A) Average number of adventitious roots (AR) in two independent
knockout alleles for gh3.3, gh3.5, and gh3.6 single mutants, in the double
mutants gh3.3-1gh3.5-2 and gh3.3-1gh3.6-1, and the triple mutant
gh3.3-1gh3.5-2gh3.6-1. Seedlings were first etiolated in the dark, until
their hypocotyls were 6 mm long, and then transferred to the light for 7 d.
Data from three independent biological replicates, each of at least 30
seedlings, were pooled and averaged. Error bars indicate SE. A one-way
analysis of variance combined with the Tukey’s multiple comparison
transcripts in the single mutants than the total GH3.3+GH3.5+GH3.6
transcript amount in the wild type (Figure 2C), which was likely to
explain the increased average number of adventitious roots in
the single mutants. The GH3.6 and GH3.5 transcripts also ac-
cumulated to higher than wild-type levels in the gh3.3-1gh3.5-2
and gh3.3-1gh3.6-1 double mutants, respectively, but this ac-
cumulation was not sufficient to be equivalent to the total GH3.3+
GH3.5+GH3.6 transcript amount in the wild type. This likely ex-
plains the reduced number of adventitious roots produced by the
gh3.3-1gh3.5-2 and gh3.3-1gh3.6-1 double mutants. In conclu-
sion, these results suggest that GH3.3, GH3.5, and GH3.6 re-
dundantly and positively regulate adventitious rooting and that this
role is independent from their activity as IAA-amido synthetases.
GH3.3, GH3.5, and GH3.6 Are Likely to Stimulate
Adventitious Rooting by Inactivating JA
The results described above suggest that GH3.3, GH3.5, and
GH3.6, which are auxin-inducible genes, do not act by regu-
lating auxin levels but might inactivate an inhibitor of adventi-
tious rooting. Among the three proteins, GH3.5 was shown to
adenylate salicylic acid (SA) as well as IAA in vitro (Staswick
et al., 2005). Therefore, it is possible that, in planta, GH3.3 and
GH3.6 proteins could also conjugate SA. Salicylate, therefore,
was an obvious candidate to test as a potential target for GH3
proteins. Like SA, JA is a stress-related hormone that can be
conjugated by GH3 proteins. The protein product of the gene
GH3.11, which is also called JASMONIC ACID RESISTANT1
(JAR1), conjugates JA to Ile, producing jasmonoyl-L-isoleucine
(JA-Ile), the active signaling molecule recognized by the F-box
receptor CORONATINE INSENSITIVE1 (COI1) (Fonseca et al.,
2009). In addition, methyl jasmonate was shown to inhibit root
growth in Arabidopsis (Staswick et al., 1992), and JA transiently
accumulated at the base of Petunia hybrida stem cuttings after
mechanical wounding but very rapidly returned to its basal level
before adventitious root formation took place (Ahkami et al.,
2009). Fattorini et al. (2009) showed that submicromolar amounts
of methyl jasmonate promoted adventitious root development from
thin cell layers of tobacco (Nicotiana tabacum). This information
posttest indicated that the differences observed in the mutants versus
the wild type (*), in gh3.6 versus gh3.3 or gh3.5 (#), and in the triple
mutant versus the double mutants (#) are significant (P < 0.01; n > 90).
Col-0, ecotype Columbia.
(B) The steady state expression levels of GH3.3, GH3.5, and GH3.6 were
quantified by quantitative RT-PCR in the hypocotyls of the different gh3
single and double mutants first etiolated in the dark, until their hypocotyls
were 6 mm long, and then transferred to the light for 72 h.
(C) The relative amount of the total GH3.3+GH3.5+GH3.6 mRNAs in the
hypocotyl of single and double gh3 mutants grown as in (B) was
quantified by quantitative RT-PCR.
For (B) and (C) Gene expression values are relative to the expression in
the wild type, for which the value is set to 1. Error bars indicate SE ob-
tained from three independent biological replicates. A one-way analysis
of variance combined with the Dunnett’s comparison posttest confirmed
that the differences between the wild type and the mutants (*) are sig-
nificant (P < 0.001, n = 3).

Figure 3. GH3.3, GH3.5, and GH3.6 Regulate Adventitious Root Initia-
tion by Modulating JA Homeostasis.
(A) to (C) Endogenous free IAA (A), free SA (B), and free JA (C) contents
were measured in the hypocotyl of wild type ecotype Columbia (Col-0) and
the gh3.3-1gh3.5-2gh3.6-1 triple mutant. Seedlings were first etiolated in
the dark, until their hypocotyls were 6 mm long (T0), and then transferred to
the light for 72 h (T72L). Error bars indicate SD of eight biological replicates.
A one-way analysis of variance combined with the Bonferroni posttest
indicated that the values indicated by asterisks are significantly different
from wild-type Col-0 values and the values indicated by hash signs are
significantly different from values observed at T0 (P < 0.05; n = 8). FW, fresh
weight.
(D) and (E) Endogenous free JA (D) and JA-Ile (E) were measured in the
hypocotyl of wild type Col-0 and the gh3.3-1gh3.5-2gh3.6-1 triple mu-
tant, etiolated as in (A) (T0) or transferred to the light for 3 h (T3L) or 9 h
(T9L). Error bars indicate SD of nine biological replicates. A one-way
analysis of variance combined with the Bonferroni posttest indicated that
the values indicated by asterisks are significantly different from wild-type
Col-0 values (P < 0.05; n = 9).
prompted us to also investigate the role of JA in adventitious
root formation on Arabidopsis hypocotyls.
To verify whether or not the reduced number of adventitious
roots observed in the gh3.3-1gh3.5-2gh3.6-1 triple mutant could
be due to a modification of endogenous SA or JA content in the
early stages of adventitious root formation, we quantified free IAA,
free JA, and free SA in hypocotyls of wild-type seedlings and
the gh3.3-1gh3.5-2gh3.6-1 triple mutant seedlings at time T0
(i.e., etiolated seedlings prior to transfer to the light) and after
transfer to the light for 72 h (T72L). No significant differences in the
free IAA or free SA content could be observed in the gh3.3-
1gh3.5-2gh3.6-1 triple mutant compared with the wild type at T0
or T72L (Figures 3A and 3B). Both free IAA and free SA contents
increased after transfer to the light in both the wild type and the
gh3.3-1gh3.5-2gh3.6-1 triple mutant. In contrast, dark-grown
seedlings of the gh3.3-1gh3.5-2gh3.6-1 triple mutant accumulated
IAA and JA Control Adventitious Rooting 2519
twice as much JA compared with the wild type (Figure 3C). Three
days after transfer to the light, the JA level was significantly re-
duced in both wild-type seedlings and the triple mutant. This result
indicates that light regulates JA homeostasis. Whether this is due
to a downregulation of JA biosynthesis or an activation of JA
degradation remains to be determined.
We had previously shown that the early events of adventitious
root initiation, such as the expression of the cell cycle gene CY-
CLIN B1, take place sometime between T0 and 48 h after transfer
to the light (Sorin et al., 2005). Therefore, we tested whether
GH3.3, GH3.5, and GH3.6 could play a role in the regulation of JA
homeostasis by quantifying free JA and the active conjugate JA-
Ile in the hypocotyl of wild type and gh3.3-1gh3.5-2gh3.6-1 triple
mutant seedlings at T0 and soon after transfer to the light (Figures
3D and 3E). JA content was significantly increased in the triple
mutant compared with the wild type, whereas that of JA-Ile was
also higher in the triple mutant but was only at a statistically sig-
nificant level 9 h after transfer to the light. Although low, this in-
crease in endogenous JA-Ile might explain the decreased number
of adventitious roots observed in the triple gh3 mutant, since
concentration as low as 1 nM exogenously applied JA reduced
by 50% the average number of adventitious roots in wild-type
seedlings (see Supplemental Figure 6 online).
In conclusion, the accumulation of JA in the triple
gh3.3gh3.5gh3.6 mutant suggests that GH3.3, GH3.5, and GH3.6
proteins might conjugate JA to amino acids whose identities have
yet to be determined, thereby modulating its level or inactivating it.
GH3.3, GH3.5, and GH3.6 Proteins May Control JA
Homeostasis by Conjugating It to Amino Acids
To establish whether GH3.3, GH3.5, and GH3.6 can conjugate JA,
end-point assays with the respective glutathione S-transferase
(GST) fusion proteins were done, and the products were ana-
lyzed by thin layer chromatography. These enzymes conjugate
IAA to several amino acids in vitro, but Asp, Met, and Trp are
among the better substrates and therefore were tested with JA
(Staswick et al., 2005). All three enzymes produce products con-
sistent with JA-Asp, JA-Met, and JA-Trp (Figure 4). In contrast, no
detectable product was seen with Ile, in agreement with the fact
that relatively little JA-Ile is produced with these enzymes. Our
data show that GH3.3, GH3.5, and GH3.6 are able to conjugate JA
to amino acids; therefore, the accumulation of JA in the gh3.3-
1gh3.5-2gh3.6-1 triple mutant might be explained by a reduction
of the conjugation process.
Nevertheless, because an accumulation of JA could also be
due to an upregulation of the biosynthesis pathway, we checked
the expression of genes encoding the main enzymes involved in
JA biosynthesis (see Supplemental Figure 7 online). All genes
tested were upregulated in the triple mutant compared with the
wild type. JA accumulation in the triple mutant could be due to
a combination of increased biosynthesis and reduced conjugation.
Jasmonate Regulates Adventitious Rooting through the
COI1 Signaling Pathway
The higher level of JA-Ile that accompanied the increase of JA
might activate the COI1 signaling pathway and thereby inhibit
2520 The Plant Cell
Figure 4. GH3.3, GH3.5, and GH3.6 Can Conjugate JA to Amino Acids in Vitro.
Thin layer chromatography analysis of JA-amino acid conjugates synthesized by recombinant GST:GH3.3, GST:GH3.5, and GST:GH3.6 proteins.
Substrates added to each enzyme reaction mixture are indicated below the image. Concentrations for JA and IAA were 10 and 1 mM, respectively. JA
conjugate standards, shown to the right, migrated with somewhat greater mobility because they were loaded on the plate in organic solvent rather than
the enzyme reaction mixture. Free IAA and JA migrated to near the top of the plate.
the early stages of adventitious root initiation. To confirm that
the JA signaling pathway was upregulated, we analyzed the ex-
pression of JAR1, JAZ1, JAZ3, JAZ5, and COI1 genes in the
hypocotyl of the wild type and the triple gh3.3-1gh3.5-2gh3.6-1
mutant. JAR1 was upregulated in the hypocotyl of the gh3.3-
1gh3.5-2gh3.6-1 mutant compared with wild-type seedlings after
they had been transferred to the light for 3 and 9 h (Figure 5A). The
relative transcript amounts of the three JA-inducible genes JAZ1,
JAZ3, and JAZ5 were upregulated in the hypocotyl of the triple
mutant whether it was transferred to the light or not (Figures 5B to
5D), and last, the relative transcript amount of COI1 was upre-
gulated in the triple mutant hypocotyl 9 h after transfer to the light
(Figure 5E). We also looked at the expression pattern of prom-
JAZ1:GUS in wild-type seedlings grown either in the dark or
transferred to the light for 24, 48, or 72 h (see Supplemental Figure
8 online). promJAZ1:GUS was expressed in the hypocotyl and
root of seedlings either kept in the dark or transferred to the light.
In seedlings transferred to the light for 72 h, the expression was
restricted to the stele. No staining was observed in the adventi-
tious root primordia (see Supplemental Figure 8C online). This
expression pattern overlapped with that of promARF6:GUS, pro-
mARF8:GUS, and promARF17:GUS, which we have described
previously (Gutierrez et al., 2009).
To further confirm that JA and the COI1 signaling pathways
were negatively regulating adventitious root development, we
analyzed the phenotypes of several mutants affected in JA bio-
synthesis and signaling (Figure 5F). We analyzed two mutants
altered in JA biosynthesis, opr3/dde1 (Sanders et al., 2000; Stintzi
and Browse, 2000) and dde2-2 (von Malek et al., 2002). Among
the T-DNA insertion lines publicly available, we identified a new
knockout mutant allele in the GH3.11/JAR1 gene that we called
jar1-12 and a GH3.11/JAR1-overexpressing line, JAR1-OX. We
also analyzed the coi1-16 allele (Ellis and Turner, 2002) altered in
the expression of COI1, the jai1/myc2 mutant (Lorenzo et al.,
2004) altered in the expression of MYC2, a bHLH transcription
factor that is a direct target of JAZ repressors and part of the core
signaling module in the jasmonate signaling pathway (Chini et al.,
2007), a 35S:MYC2 line, the triple mutant myc2myc3myc4 that is
as impaired as coi1-1 in the activation of several, but not all, JA-
mediated responses (Fernández-Calvo et al., 2011), and last, the
JA-insensitive mutant jai3-1 altered in the expression of JAZ3 (Chini
et al., 2007). These mutants were characterized under the same
conditions used previously (Figure 2A). Both opr3/dde1 and dde2-
2, which are affected in JA biosynthesis, developed more adven-
titious roots than the wild type, confirming a negative effect of JA on
adventitious root development (Figure 5F). Interestingly, two SA-
deficient mutants, eds5-1 and eds5-2 (Rogers and Ausubel, 1997),
that we also analyzed produced fewer adventitious roots than did
the wild type (Figure 5G), suggesting that SA is possibly a positive
regulator of adventitious rooting. The jasmonate-insensitive jai3-1
mutant did not show any difference in the average number of ad-
ventitious roots compared with the wild type, whereas lines in
which the JA signaling pathway was upregulated (JAR1-OX and
35S:MYC2) had significantly fewer adventitious roots than the wild
type, and those lines with a downregulated JA signaling pathway
(jar1-12, coi1-16, myc2, and myc2myc3myc4) all had more ad-
ventitious roots than the wild type (Figure 5F). Noticeably, coi1-16
and the myc2myc3myc4 triple mutant developed the highest av-
erage number of adventitious roots, confirming that the COI1 sig-
naling pathway plays a role in the control of adventitious rooting.
The upregulated JA signaling pathway in the hypocotyl of the
gh3.3-1gh3.5-2gh3.6-1 mutant explains the reduced number of
adventitious roots, since the double mutants arf6-3coi1-16, arf8-
7coi1-16, and coi1-16ARF17-OX developed more adventitious
roots than the wild type, whereas the single arf mutants and the
ARF17-OX line had fewer (Figure 5H). This last result confirms that
JA negatively regulates adventitious root initiation through COI1,
which acts downstream of the ARF/GH3 module.
DISCUSSION
In a previous study, we showed that adventitious rooting on the
Arabidopsis hypocotyl is controlled by a complex interaction be-
tween three transcription factors from the ARF family, ARF6, ARF8,

Figure 5. The COI1 Signaling Pathway Is Required for the Inhibition of
Adventitious Rooting by Jasmonate.
IAA and JA Control Adventitious Rooting 2521
and ARF17, and the regulatory microRNAs miR167 and miR160.
ARF6 and ARF8 are positive regulators of adventitious rooting,
whereas ARF17 is a negative regulator (Gutierrez et al., 2009). In
addition, the levels of the proteins encoded by three auxin-re-
sponsive genes from the GH3 family (GH3.3, GH3.5, and GH3.6)
were positively correlated with the number of adventitious roots,
suggesting a role for these genes in the regulation of adventitious
rooting (Sorin et al., 2006).
The GH3 family is part of the broader acyl-adenylate/thioester-
forming enzyme family, also called the firefly luciferase family
(Staswick et al., 2002). In Arabidopsis, there are 19 members
that fall into three distinct phylogenetic groups (Staswick et al.,
2002, 2005). In group I, GH3.10/DFL2 was described as being
involved in red light-specific hypocotyl elongation (Takase
et al., 2004), and GH3.11/JAR1 was first shown to be required
for the root growth inhibition response to methyl jasmonate
(Staswick et al., 1992) and later to catalyze the formation of JA-Ile,
the bioactive form of JA (Staswick and Tiryaki, 2004). In addition to
GH3.3, GH3.5, and GH3.6/DFL1, group II of the GH3 family
comprises five additional members that catalyze the formation
of auxin-amino acid conjugates (Staswick et al., 2005). And
last, group III is so far the least well characterized. It is composed
of nine proteins, one of which (GH3.12/PBS3) has recently been
shown to conjugate amino acids to 4-substituted benzoates
(Okrent et al., 2009). We analyzed the relative transcript amount of
all GH3 genes and a few additional auxin-responsive genes, and
with the exception of GH3.3, GH3.5, and GH3.6/DFL1, it was not
correlated with the adventitious root phenotype of the arf mutants
and ARF-overexpressing lines. Transcripts were either not detected
(A) to (E) Quantification by quantitative RT-PCR of JAR1 (GH3.11) JAZ1,
JAZ3, JAZ5, and COI1 transcripts in hypocotyls of wild-type etiolated
seedlings (T0), after an additional 3 or 9 h in the dark (T3D or T9D), and
after transfer to the light for 3 or 9 h (T3L or T9L). Values are relative to the
expression level of APT1, which was used as a reference gene as de-
scribed in Methods. Error bars indicate SE obtained from three independent
biological replicates. A one-way analysis of variance combined with the
Tukey’s multiple comparison posttest confirmed that the differences be-
tween the wild type and the mutants (*) are significant (P < 0.05, n = 3).
Col-0, ecotype Columbia.
(F) and (G) Average number of adventitious roots (AR) in several mutants
altered in jasmonate biosynthesis or signaling (F) and in the SA-deficient
mutant lines eds5-1 and eds5-2 (G). Data from three independent bio-
logical replicates, each of at least 30 seedlings, were pooled and averaged.
Error bars indicate SE. A one-way analysis of variance combined with the
Tukey’s multiple comparison test showed that the values indicated by
asterisks are significantly different from wild-type values (P < 0.01; n > 90);
the Bonferroni posttest indicates that the value indicated by the plus sign is
significantly different from that of the myc2 mutant and that indicated by
the hash mark is significantly different from that of jar1-12 (P < 0.01; n > 90).
(H) Average number of adventitious roots in single arf6-3 and arf8-7
mutants and the ARF17-OX line and in the corresponding double
mutants with coi1-16. Data from three independent biological repli-
cates, each of at least 30 seedlings, were pooled and averaged. Error
bars indicate SE. A one-way analysis of variance combined with the
Dunnett’s multiple comparison test showed that the values indicated
by asterisks are significantly different from wild-type values (P < 0.05;
n > 90).
2522 The Plant Cell
Figure 6. Adventitious Root Initiation: A Proposed Model for a Regula-
tory Pathway.
ARF6, ARF8, and ARF17 and their regulatory microRNAs interact in
a complex network (Gutierrez et al., 2009) and act upstream of GH3.3,
GH3.5, and GH3.6. ARF6 and ARF8 are positive regulators of the three
GH3s, whereas ARF17 is a negative regulator. GH3.3, GH3.5, and GH3.6
control each other’s expression through a pathway yet to be identified.
The three GH3 genes control JA homeostasis. The JA-Ile level in the
hypocotyl is determined by the JA level. The level of JA-Ile negatively
regulates adventitious rooting through the activation of the COI1 sig-
naling pathway. In conclusion, auxin controls adventitious root initiation
through the activation of ARF6 and ARF8 proteins, leading to a down-
regulation of the inhibiting COI1 signaling pathway.
in Arabidopsis hypocotyls or their level was either not affected
or significantly increased in the different arf mutants or ARF-
overexpressing lines. We could then conclude that overall
auxin signaling was not affected in the arf mutants and ARF-
overexpressing lines and that GH3.3, GH3.5, and GH3.6 are
the only GH3 genes out of 19 that are strictly coregulated in
the Arabidopsis hypocotyl during adventitious rooting. Fur-
thermore, a decrease or increase in the GH3 transcript level leads
to a lower or higher average number of adventitious roots,
respectively, confirming the positive correlation we described
previously (Sorin et al., 2006).
Exogenous auxin is often used to induce adventitious rooting
on stem cuttings of different species (de Klerk et al., 1999; Geiss
et al., 2009; Li et al., 2009). In Arabidopsis, mutants overproducing
auxin, such as superroot1, superroot2, or yucca1, spontaneously
develop numerous adventitious roots on the hypocotyl and, in the
case of superroot1, on cuttings of different organs (Boerjan et al.,
1995; Delarue et al., 1998; Zhao et al., 2001). Nevertheless, in this
work, we show that the variation in the average number of ad-
ventitious roots developing on the hypocotyl of representative arf
mutants, ARF-overexpressing lines, and the gh3.3, gh3.5, and
gh3.6 single and triple mutants does not correlate with a change in
the free IAA content. Rather, the data are consistent with our
previous results showing that the number of adventitious roots is
correlated to the level of GH3.3, GH3.5, and GH3.6 proteins (Sorin
et al., 2005, 2006). This was unexpected, given the evidence that
these GH3s inactivate IAA by conjugating it to amino acids
(Staswick et al., 2005). Consistent with this function, the levels of
IAGlu and IAAsp were reduced in the single gh3.3-1, gh3.5-2, and
gh3.6-1 mutant hypocotyls compared with the wild type. How-
ever, this did not increase the endogenous level of free IAA.
Surprisingly, in each single mutant, the other two GH3 genes were
upregulated, so one might actually have predicted a reduction of
free IAA and a reduced number of adventitious roots, which did
not occur. Together, these results suggest that these three GH3
proteins are not directly regulating IAA homeostasis in hypocotyls
in a way that controls adventitious rooting.
The single gh3 mutants developed more adventitious roots
than the wild type likely because of the upregulation of the other
two remaining GH3 genes, which resulted in a total amount of
the three GH3 transcripts that was higher in the single mutants
than the total amount in the wild type. In contrast, the double
knockout mutants (gh3.3-1gh3.5-2 and gh3.3-1gh3.6-1) de-
veloped fewer adventitious roots compared with the wild type,
and the gh3.3-1gh3.5-2gh3.6-1 triple mutant developed even
fewer than the double mutants. This additive effect indicates
that the GH3.3, GH3.5, and GH3.6 genes have redundant
functions, but none of them alone can fully compensate for the
lack of expression of the other two.
These results indicate that ARF6, ARF8, and ARF17 transcrip-
tion regulators and their downstream GH3 targets do not control
adventitious rooting by modulating free IAA content. Rather,
GH3.3, GH3.5, and GH3.6 appear to be promiscuous enzymes
that affect the homeostasis of one or more other compounds that
negatively regulate adventitious rooting.
Because GH3.5 was described as acting at the crosstalk of
auxin homeostasis and SA signaling (Zhang et al., 2007, 2008), we
investigated whether SA was an inhibitor of adventitious rooting.
The two mutant alleles eds5-1 and eds5-2, which are deficient in
SA biosynthesis (Rogers and Ausubel, 1997), produced fewer
adventitious roots than the wild type, suggesting that SA was not
a negative, but likely a positive, regulator of adventitious rooting.
Furthermore, free IAA and SA contents were not altered in the
triple gh3 mutant compared with the wild type, either in the dark or
72 h after transfer to the light, although in both genotypes the IAA
and SA contents significantly increased after transfer to the light.
Thus, the phenotype of the triple gh3 mutant was not due to
a modification in the level of free IAA or SA.
In contrast with SA mutants, JA-deficient mutants opr3/dde1
(Sanders et al., 2000; Stintzi and Browse, 2000) and dde2-2 (von
Malek et al., 2002) produced more adventitious roots than the
wild type, suggesting that jasmonate is an inhibitor of adventi-
tious rooting. Free JA in the hypocotyl of the gh3.3-1gh3.5-
2gh3.6-1 triple mutant was twice that in the wild type at T0, and
after 3 and 9 h in the light, the percentage of decline in en-
dogenous JA was greater for the triple mutant than for the wild
type, but it was still significantly higher than in the wild type at 9
h. Three days after transfer to the light, the amount was mark-
edly decreased in both the wild type and the triple mutant, in
agreement with published results showing that JA biosynthesis
is regulated by light (Zhai et al., 2007). One way to explain the

higher JA levels in the triple gh3 mutant is if the three GH3 en-
zymes regulate JA homeostasis by conjugating it to amino
acids, leading either to JA catabolism or its unavailability for
synthesis of the JA-Ile signal by GH3.11/JAR1. All three en-
zymes produced JA conjugates in vitro with three of the amino
acids tested in this study, but importantly, none synthesized
appreciable amounts of JA-Ile. The JA concentration used in the
enzyme assays here was 10-fold higher for JA than for IAA,
because previous studies indicated that GH3.3 and GH3.5 were
considerably less active on JA (Staswick et al., 2002). Never-
theless, the results suggest that JA could be an alternative
substrate for GH3.3, GH3.5, and GH3.6, pointing to a role in
controlling the level of free JA at least in hypocotyl tissues. In
this study, JA-Trp was not detected in the hypocotyl of the wild
type or gh3.3-1gh3.5-2gh3.6-1. JA-Trp is an inhibitor of auxin
responses, and we previously found low levels in Arabidopsis
(Staswick, 2009). In any case, the auxin-inhibiting activity of JA-
Trp is not likely responsible for regulating adventitious rooting,
because the latter is COI1 dependent whereas JA-Trp activity is
COI1 independent. JA-Met and JA-Asp have not been reported
to occur in Arabidopsis, but even if synthesized, they may not
accumulate appreciably if they are further metabolized, as is the
case for IAAsp.
We also observed that the expression of genes encoding
enzymes involved in JA biosynthesis was upregulated in the
triple mutant, suggesting that the increase of JA could be due to
an upregulation of its biosynthesis. We cannot at present dis-
criminate between these two hypotheses, and indeed, the
increase in JA might result from a combination of decreased
conjugation and increased biosynthesis in the triple gh3 mutant.
The elevated JA content was accompanied by a small increase
in JA-Ile at 9 h after transfer to the light, and this coincided with
the upregulation of the GH3.11/JAR1 transcript. Several JA
biosynthesis genes are well established to be transcriptionally
upregulated by JA-Ile/COI1 signaling, so reduced conjugation of
JA to other amino acids in the triple mutant might free up more
JA for JA-Ile synthesis, which in turn could enhance JA pro-
duction. This is consistent with a model that includes JA-Ile
signaling as a negative regulator of adventitious rooting.
JA-Ile promotes the interaction of the F-box receptor protein
COI1 with the JAZ coreceptors, which also function as transcrip-
tional repressors of jasmonate response genes (Thines et al.,
2007). JAZ proteins are then ubiquitinated, targeting them for
degradation by the 26S proteasome, followed by the activation of
associated JA-dependent responses (Chini et al., 2007; Yan et al.,
2007; Chung et al., 2008). Further evidence that JA-Ile/COI1 sig-
naling negatively regulates adventitious rooting is the finding that,
with the exception of the jasmonate-insensitive jai3-1 mutant, all of
the tested downregulation mutants in this pathway (jar1-12, coi1-
16, myc2, and myc2myc3myc4) developed significantly more ad-
ventitious roots than the wild type, while the upregulation lines
(JAR1-OX and 35S:MYC2) were deficient in root production. The
lack of a phenotype in jai3-1 could be explained if this particular
JAZ protein (JAZ3) is not involved in adventitious rooting or if other
JAZ proteins are redundant for its activity. The JAZ1, JAZ3, and
JAZ5 transcripts were elevated in the gh3.3-1gh3.5-2gh3.6-1 triple
mutant kept in the dark or transferred to the light, indicating that
jasmonate signaling was indeed upregulated in the hypocotyl at
IAA and JA Control Adventitious Rooting 2523
this time. The COI1 transcript was also higher in the hypocotyl of
the triple mutant 9 h after transfer to the light. The increase in
adventitious roots in the double mutants coi1-16arf6-7, coi1-
16ar8-7, and coi1-16ARF17-OX, in contrast to the reduced rooting
in the single arf6-3, arf8-7, and ARF17-OX lines, supports the idea
that the COI1-dependent jasmonate signaling pathway acts down-
stream of the regulatory module composed of three ARFs (ARF6,
ARF8, and ARF17) and the three GH3s they regulate (GH3.3, GH3.5,
and GH3.6).
In conclusion, our data strongly support the hypothesis that
the JA-Ile/COI1 signaling pathway negatively regulates adven-
titious rooting in Arabidopsis hypocotyls. We propose that auxin
stimulates adventitious rooting by inducing GH3.3, GH3.5, and
GH3.6 gene expression, via the positive regulators ARF6 and
ARF8, leading to an increase in JA conjugation and, as a con-
sequence, a reduction in free JA level. These interactions can be
added to our previous model that integrated the interactions
between the three ARFs and their regulatory microRNAs
(Gutierrez et al., 2009) and are summarized in the model presented
in Figure 6. ARF6 and ARF8 have previously been described as
positive regulators of JA biosynthesis during flower development
(Nagpal et al., 2005; Tabata et al., 2010); here, we show that they
are likely to regulate the conjugation of jasmonate to amino acids.
Our results highlight a regulatory pathway at the crosstalk of IAA
and JA, in which ARF6, ARF8, and ARF17 and their downstream
targets GH3.3, GH3.5, and GH3.6 are involved, and demonstrate
that JA homeostasis is under auxin control.
METHODS
Plant Material and Growth Conditions
The ARF6-OX, ARF8-OX, and ARF17-OX1 lines have been described
previously (Nagpal et al., 2005). Line MIR160c-OX, which overexpresses
the gene MIR160c, was described by Wang et al. (2005). The knockout
mutants arf8-7 and arf6-3 were described by Gutierrez et al. (2009). The
SA-deficient mutant lines eds5-1 and eds5-2 were obtained from Fred-
erick Ausubel. Seeds from the two JA-deficient mutants dde2-2 and opr3
were gifts from Beate Keller and John Browse, respectively. Seeds from
coi1-16, jai3-1, myc2, 35S:MYC2, and the myc2myc3myc4 triple mutant
were gifts from Laurens Pauwels.
Insertion lines for the GH3.3, GH3.5, GH3.6, and GH3.11/JAR1 genes
were obtained from the Nottingham Arabidopsis Stock Centre. The
presence of each insertion was verified using the gene-specific primers
listed in Supplemental Table 1 online. The absence or the overexpression
of a full-length mRNA was verified for each line by RT-PCR using primers
for the coding region (see Supplemental Table 2 online). We identified two
gh3.3 mutant alleles, gh3.3-1 (SM_3-39271) and gh3.3-2 (SM_3-17798);
two gh3.5 mutant alleles, gh3.5-1 (SALK_014376) and gh3.5-2 (SALK_
151766), which was also described by Zhang et al. (2007); two gh3.6
mutant alleles, gh3.6-1 (SALK_133707) and gh3.6-2 (SALK_060813);
one gh3.11 mutant allele (SALK_011510) that we called jar1-12 according to
Suza and Staswick (2008); and a GH3.11-overexpressing line, JAR1-OX
(SALK_013425).
For generating double mutants with arf6-3, arf8-7, and ARF17-OX1
lines, the coi1-16 allele described by Ellis and Turner (2002) was used. In
the F2 population, homozygous arf6-3, arf8-7, and ARF17-OX1 were
identified by PCR using the genotyping primers listed in Supplemental
Table 1 online. To genotype the coi1-16 point mutation, new derived
cleaved-amplified polymorphic sequence primers were designed using
the dCAPS Finder 2.0 software (Neff et al., 2002; http://helix.wustl.edu/
2524 The Plant Cell
dcaps/dcaps.html). A mismatch (underlined) was introduced in the F
primer to incorporate a restriction site in the PCR product of one allele
(see Supplemental Table 1 online). After amplification, the PCR products
were digested with XbaI (Fermentas Fast Digest) following the manu-
facturer’s recommendations and separated on a 4% agarose gel. The
wild-type allele yielded two fragments of 146 and 19 bp, while coi1-16
gave one band of 165 bp.
Adventitious rooting experiments were performed as described pre-
viously (Gutierrez et al., 2009).
Expression Profiling of GH3 Genes
A 1.5-kb-long fragment upstream from the start codon of GH3.3, GH3.5,
and GH3.6 was amplified from genomic DNA by PCR, cloned using
a pENTR/D-TOPO Cloning Kit (Invitrogen), and transferred into the
pKGWFS7 binary vector (Karimi et al., 2002) using a Gateway LR Clonase
Enzyme Mix (Invitrogen) according to the manufacturer’s instructions.
Transgenic Arabidopsis thaliana plants expressing the different promGH3:
GUS fusions were produced, and the expression pattern was checked in the
T2 progeny of 10 to 15 independent transgenic lines. One representative
homozygous line for each gene was used for further characterization.
Histochemical assays for GUS expression were performed as described
previously (Sorin et al., 2005).
RNA Isolation and cDNA Synthesis
RNAs from arf and gh3 mutants and ARF-overexpressing lines were
prepared as described by Gutierrez et al. (2009). Portions (5 mg) of the
resulting RNA preparations were treated with DNaseI using a DNAfree Kit
(Ambion), and cDNA was synthesized by reverse transcribing 5 mg of total
RNA using SuperScript III reverse transcriptase (Invitrogen) with 2.5 mg of
random hexamers and 500 ng of oligo(dT)12 primer according to the
manufacturer’s instructions. The reaction was stopped by incubation at 70°
C for 10 min; the reaction mixture was then treated with RNaseH (Invitrogen)
according to the manufacturer’s instructions and diluted by adding 1 mL of
distilled water. All cDNA samples were tested by PCR using specific primers
flanking an intron sequence to confirm the absence of genomic DNA
contamination.
Real-Time RT-PCR Experiments
Transcript levels for Figures 1 and 2 were assessed by quantitative RT-PCR
as described by Gutierrez et al. (2009). Transcript levels for Supplemental
Figures 2 and 3 online were assessed by quantitative RT-PCR, in assays
with triplicate reaction mixtures (final volume, 20 mL) containing 5 mL of
cDNA, 0.3 mM of both forward and reverse primers, and 13 FastStart DNA
MasterPLUS SYBR Green I (Roche), using a Roche LightCycler, and crossing
threshold (CT) values for each sample were acquired with LightCycler
software 3.5 (Roche) using the second derivative maximum method.
Transcript levels for Figure 5 and Supplemental Figure 7 online were as-
sessed by quantitative RT-PCR, in assays with triplicate reaction mixtures
(final volume, 10 mL) containing 2 mL of cDNA, 0.5 mM of both forward and
reverse primers, and 13 LightCycler 480 SYBR Green I Master (Roche) on
384-well plates. The plates were filled by an EVO150 robotic liquid handling
platform (Tecan), and quantitative PCR was performed with a LightCycler
480 (Roche). The CT values for each sample were acquired with the
LightCycler 480 software (Roche) using the second derivative maximum
method. All quantifications were repeated with three independent biological
replicates.
Steady state levels of uncleaved ARF transcripts were quantified using
primers spanning the microRNA target site. The following standard
protocol was applied for the amplification of each mRNA: 10 min at 95°C,
followed by 40 cycles of 10 s at 95°C, 15 s at 60°C (except for GH3.5, for
which the annealing temperature was 65°C), and 15 s at 72°C. The
sequences of primers used for all target genes are presented in
Supplemental Table 2 online. Due to the high sequence conservation, note
that the primers for GH3.13 might also produce an amplicon from GH3.15
transcripts, which was not a problem in our analysis since GH3.15
transcripts are not detectable.
Each amplicon was first sequenced to ensure the specificity of the
amplified sequence, and in order to check that the fluorescence signal
was derived from the single intended amplicon in the following runs,
a melting curve analysis was added to each PCR program.
Real-Time RT-PCR Data Analysis
APT1 and TIP41 had previously been validated as the most stably
expressed genes among 11 tested in our experimental procedures
(Gutierrez et al., 2009) and were used to normalize the real-time RT-PCR
data. The normalized expression patterns obtained using both reference
genes were similar, so only the data normalized with APT1 are shown. For
Figures 1 and 2 and Supplemental Figure 3 online, CT and PCR effi-
ciency (E) values were used to calculate expression using the formula
ET(CTWT-CTM)/ER(CTWT-CTM), where T is the target gene and R is the ref-
erence gene, CT is the crossing threshold value, M refers to cDNA from
the mutant line, and WT refers to cDNA from the wild type. In these
figures, the data for mutants are presented as relative to the wild type,
the calibrator. For Figure 5 and Supplemental Figures 2 and 7 online, the
expression in the wild type and/or mutants was calculated using the formula
ERCTWT/ETCTWT [i.e., (1/ETCTWT)/(1/ERCTWT): the normalized relative quantity of
template in the original sample], the expression levels of target genes being
relative to those of the reference gene. All real-time RT-PCR results presented
are means from three independent biological replicates. For each in-
dependent biological replicate, the relative transcript amount was calculated
as the mean of three technical replicates, using the method for calculation of
SE values in relative quantification recommended by Rieu and Powers (2009).
IAA and IAA Conjugate Analysis
Seedlings from each genotype were grown in vitro as described above.
Seventy-two hours after transfer to the light, cotyledons and roots were
removed, and an average of 150 hypocotyls (50 to 70 mg) per replicate
were harvested, dried on tissue paper, and frozen in liquid nitrogen. Up to
nine replicates from three independent experiments were harvested.
Samples were extracted, purified, and analyzed by liquid chromatogra-
phy–multiple reaction monitoring–mass spectrometry as described pre-
viously (Kowalczyk and Sandberg, 2001).
Free IAA, JA, JA-Ile, and SA Quantification
Hypocotyls from the wild type and triple gh3 mutants were collected as
described above. JA, JA-Ile, SA, and IAA extraction, purification, and
quantification were performed according to Bergougnoux et al. (2009),
with modifications. To each extract, 20 pmol of [2H6]JA and [2H2]JA-Ile
(Olchemim) and 100 pmol of [2H4]SA and [13C6]IAA (Cambridge Isotope
Laboratories) were added as internal standards to check recovery during
purification and to validate the quantification. Purified samples were
analyzed by the liquid chromatography–tandem mass spectrometry
system consisting of an ACQUITY UPLC System (Waters) and a Xevo TQ
(Waters) triple quadrupole mass spectrometer. Samples were dissolved in
15% acetonitrile, injected onto the ACQUITY UPLC BEH C18 column (100
3 2.1 mm, 1.7 µm; Waters), and eluted with a linear gradient (0 to 3 min,
15% B; 3 to 10 min, 20% B; 10 to 20 min, 30% B; flow rate of 0.25 mL/min)
of 7.5 mM formic acid (A) and acetonitrile (B). Quantification was obtained
using a multiple reaction monitoring mode of [M-H]+/[M-H]2 and the
appropriate product ion. The multiple reaction monitoring transitions
215.1>59, 326.2>151.1, 141.1>97, and 182.1>136.1 were used for la-
beling, and 209.1>59, 324.2>151.1, 137.1>93, and 176.1>130.1 were

used for authentic JA, JA-Ile, SA, and IAA, respectively. For selective
multiple reaction monitoring experiments, optimal conditions were as
follows: capillary/cone voltage, 1.0 kV/25 V; source/desolvation gas
temperature, 120/550°C; cone/desolvation gas, 70/1000 L/h; collision
gas, 0.21 mL/min; low mass/high mass resolution, 2.8/12.5; collision
energy, 14 eV; ion energy 1/2, 1.0/0.7 V; entrance/exit, 2.0 V. The limits of
detection (signal-to-noise ratio of 1:3) for all analytes were close to 50.0
fmol. The linear range was at least over the 5 orders of magnitude with
a correlation coefficient of 0.9989 to 0.9998. For each mutant, three in-
dependent experiments were performed, each composed of three bi-
ological replicates.
Enzymatic Assays
Enzyme assays were done using GH3-GST fusion proteins produced in
Escherichia coli as described previously (Staswick et al., 2005). Reactions
were for 16 h at 23°C in 50 mM Tris-HCl, pH 8.6, 1 mM MgCl2, 1 mM ATP,
1 mM DTT, and 2 mM amino acid. Either IAA (1 mM) or JA (10 mM) was
included in each reaction. Reactions were analyzed on silica gel 60 F260
plates developed in chloroform:ethyl acetate:formic acid (35:55:10, v/v)
and then stained with vanillin reagent (6% vanillin [w/v], 1% sulfuric acid
[v/v] in ethanol).
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession num-
bers: ACX1 (At4g16760) AIR12 (At3g07390), AOC2 (At3g25770), AOS
(At5g42650) APT1 (At1g27450), ARF6 (At1g30330), ARF8 (At5g37020), ARF17
(At1g77850), COI1 (At2g39940), GH3.1 (At2g14960), GH3.2 (At4g37390),
GH3.3 (At2g23170), GH3.4 (At1g59500), GH3.5 (At4g27260), GH3.6
(At5g54510), GH3.7 (At1g23160), GH3.8 (At5g51470), GH3.9 (At2g47750),
GH3.10 (At4g03400), GH3.11 (At2g46370), GH3.12 (At5g13320), GH3.13
(At5g13350), GH3.14 (At5g13360), GH3.15 (At5g13370), GH3.16 (At5g13380),
GH3.17 (At1g28130), GH3.18 (At1g48670), GH3.19 (At1g48660), GH3.20
(At1g48690), IAA7 (At3g23050), IAA17 (At1g04250), IAA19 (At3g15540), IAA28
(At5g25890), JAZ1 (At1g19180), JAZ3 (At3g17860), JAZ5 (At1g17380), KAT2
(At2g33150), LOX2 (At3g45140), OPCL1 (At1g20510), OPR3 (At2g06050), and
TIP41 (At4g34270).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Content of IAA and Its Major Conjugates in
Hypocotyls of the Wild Type, arf Mutants, and ARF-Overexpressing Lines.
Supplemental Figure 2. Expression Pattern of GH3.3, GH3.5, and
GH3.6.
Supplemental Figure 3. Relative Amount of the Sum of GH3.3,
GH3.5, and GH3.6 mRNAs in the Single arf6-3 and arf8-7 Mutants and
the arf6-3arf8-7(+/2) Sesquimutant.
Supplemental Figure 4. Root Length and Lateral Root Number in gh3
Single, Double, and Triple Mutants.
Supplemental Figure 5. Content of IAA and Its Major Conjugates in
Hypocotyls of the Wild Type and gh3.3-1, gh3.5-1, and gh3.6-1 Mutants.
Supplemental Figure 6. Effect of Exogenously Applied JA on
Adventitious Rooting.
Supplemental Figure 7. Jasmonate Biosynthesis is Upregulated in
the Triple gh3.3gh3.5gh3.6 Mutants.
Supplemental Figure 8. Expression Pattern of promJAZ1:GUS.
Supplemental Table 1. Sequences of Primers Used for Genotyping
Newly Identified gh3 Mutants and Overexpressing Lines.
IAA and JA Control Adventitious Rooting 2525
Supplemental Table 2. Sequences of Primers Used for Quantifying
Target Genes by Real-Time RT-PCR.
ACKNOWLEDGMENTS
We thank the Nottingham Arabidopsis Stock Centre for providing seeds.
We are very grateful to Kotaro T. Yamamoto (Hokkaido University), Jason
W. Reed (University of North Carolina), Xiao-Ya Chen (Shanghai Institutes
for Biological Sciences), Frederick Ausubel (Harvard Medical School),
Beate Keller (Institute of Plant Biology, University of Zurich), John Browse
(Institute of Biological Chemistry, Washington State University), and
Laurens Pauwels (Flemish Institute for Biotechnology, Department of Plant
Systems Biology, Ghent University Technologiepark) for kindly providing
mutant or overexpressing lines. This work was supported by the Swedish
Natural Sciences Research Council, the Swedish Foundation for Strategic
Research, the Swedish Research Council for Research and Innovation for
Sustainable Growth, the K.&A. Wallenberg Foundation, the Carl Trygger
Foundation, the University of Picardie Jules Verne, the Regional Council of
Picardie, the European Regional Development Fund, and the Grant Agency
of the Academy of Sciences of the Czech Republic (KAN 200380801).
AUTHOR CONTRIBUTIONS
K.F. and D.I.P. contributed equally to this work. L.G. and G.M. character-
ized the mutant phenotypes and performed the quantitative PCR analysis.
L.G., G.M., and D.I.P. performed the GUS assays. D.I.P. produced the
multiple mutants. D.I.P. and M.P. genotyped the mutants. K.F., O.N., and
M.K. performed the hormone measurements. P.S. performed the enzy-
matic assays. H.D. produced the biological material for quantitative PCR
analysis and hormone measurements. G.G. produced the prom:GUS lines.
L.G. and C.B. designed the research and analyzed the data. C.B.
conceptualized and supervised the overall project. C.B. wrote the article
with inputs from L.G., O.N., M.K., and P.S. P.S. edited the article.
Received April 13, 2012; revised May 29, 2012; accepted June 12, 2012;
published June 22, 2012.
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